Therapeutic agent for nervous system disease

ABSTRACT

An object is to provide a drug which is useful in treating a nervous system disease. A drug containing vitamin B12 as an active ingredient according to the present invention has an M2 macrophage/microglia induction promoting effect, an M1 macrophage/microglia induction inhibiting effect, a nerve regeneration promoting effect, and the like and is very useful as a therapeutic agent for a nervous system disease, and particularly useful as a therapeutic agent for a central nervous system disease such as cerebral infarction, dementia, or spinal cord injury.

This is a Division of application Ser. No. 16/907,913 filed Jun. 22,2020, which is a Bypass Continuation of International Application No.PCT/JP2018/046945 filed Dec. 20, 2018, which claims the benefit ofJapanese Application No. 2018-156503 filed Aug. 23, 2018 and JapaneseApplication No. 2018-156503 filed Dec. 21, 2017. The disclosures of theprior applications are hereby incorporated by reference herein in theirentireties.

TECHNICAL FIELD

The present invention relates to a therapeutic agent for nervous systemdisease or the like.

BACKGROUND ART

Nervous system diseases occur in the brain, spinal cord, peripheralnerves, and muscles. Among the diseases, those that affect the brain andspinal cord are referred to as central nervous system diseases.Representative examples of the central nervous system diseases occurringin the brain include cerebral infarction and dementia. Representativeexamples of the central nervous system diseases occurring in the spinalcord include spinal cord injury.

Cerebral infarction accounted for about 60 percent of cases ofcerebrovascular disorder, which was the fourth leading killer for theyear of 2014. Given that patients are very likely to need care afterhaving cerebral infarction, the disease greatly affects society in termsof healthcare cost. The zones of cerebral infarction are divided into anischemic core (also referred to simply as a core), in which blood flowis completely blocked, and a peripheral penumbra (half-shaded zone), inwhich blood flow is maintained by collateral circulation. While rescuingthe core portion, where nerve cells rapidly die (primary injury), isdifficult, the penumbral portion may survive because cells escape deathin this portion. Thus, how to rescue the penumbral portion is a vitalpoint in acute-phase treatment of cerebral infarction. Histologicalchanges in the cerebral infarction lesion include (1) apoptosis of nervecells, (2) induction of inflammation, and (3) breakdown of the bloodbrain barrier (BBB). Examples of cerebral infarction therapeutic agentscurrently used in Japan include urokinase, anticoagulation agents, andantiplatelet agents, as well as agents that dilute blood and agents thatreduce edema. Edaravone (trade name, Radicut) was approved as a drug forscavenging free radicals in Japan in 2001 but has not been approved inEurope, the United States, or the like. A thrombolytic therapy (tissueplasminogen activator [tPA] therapy) was approved in 2005, but its useis restricted to treatment given within 4.5 hours from the onset of thedisease. Then, no additional therapeutic drug for cerebral infarctionhas become available since 2005.

It is reported that there are about 5000 new patients with spinal cordinjury each year in Japan. Pain, numbness, motor dysfunction, and thelike are associated with an extremely decreased QOL of patients.Pathogenesis of spinal cord injury involves damage to nerve cells andvascular tissue due to a direct external force at the time of injury(primary injury), followed by a series of reactions associated with thebreakdown of the blood-spinal cord barrier (secondary injury), whichresults in expansion of the injured area. Since the primary injury isinevitable, how to reduce the secondary injury is critical in the acuteto subacute-phase treatment of spinal cord injury. However, no safe andeffective therapeutic drug is currently available for the treatment ofacute-phase spinal cord injury in clinical setting. The treatmentguidelines for acute spinal cord injury issued in the United Statesclearly state that conventional therapies using high-dosemethylprednisolone should not be used routinely because of their severeside effects. Thus, there is an unmet medical need for a noveltherapeutic agent effective for spinal cord injury.

Peripheral nerves have a capacity to regenerate after injury, but it isnot sufficient for restoring neural functions. When a nerve is injured,Wallerian degeneration, i.e., phagocytotic elimination of axons andmyelin sheaths, occurs. In the subsequent regeneration process, aregenerated axon extends in a distal direction along the Bungner's bandformed by undifferentiated Schwann cells, leading to reinnervation ofthe target muscle. Eventually, a myelin sheath is formed by Schwanncells which surround the regenerated axon. However, the regeneratednerve extends at a very low rate, and muscular atrophy occurs if thedistance to the target muscle is long; thus, sufficient functionalrecovery cannot be expected. In recent years, it has been found thatmacrophages play an important role in each step of the regenerationprocess, and this finding is attracting attention. While thepro-inflammatory function of macrophages is well known, there is alsoanother phenotype that has an anti-inflammatory function, opposite toit, and these two phenotypes, referred to as an M1 and an M2,respectively, are viewed as a continuum (switch between M1 and M2 canoccur). In general, it is said that nerve regeneration can be promotedby increasing M2 macrophage, which is an anti-inflammatory phenotype.

On the other hand, central nervous system is known to have a lowercapacity to regenerate compared with peripheral nervous system. It isknown that, after central nerve injury, an axon outgrowth inhibitor isexpressed in oligodendrocytes, which are cells forming a myelin sheatharound an axon, and that macrophage/microglia, astrocyte, and the likeform glial scar, which exerts an inhibitory effect on axon outgrowth. Itis therefore said that suppression of the pro-inflammatory effect ofmacrophage/microglia is important after central nerve injury, and thatnerve regeneration can be promoted by increasing M2macrophage/microglia, which is an anti-inflammatory phenotype, as withperipheral nervous system.

It is known that vitamin B₁₂ is effective for treating vitamin B₁₂deficiency, and that neurologic changes, such as peripheral neuritis orspinal cord change, may occur in vitamin B₁₂ deficiency (Patent Document1).

In addition, patients with cerebral ischemia have an elevated bloodlevel of homocysteine as compared with healthy individuals, suggestingan association between the blood homocysteine level and cerebralischemia. Further, it has been found that the blood homocysteine levelis decreased by administration of folic acid, vitamin B₆, and vitaminB₁₂, suggesting that decreases in the homocysteine level can potentiallyreduce the risk of cerebral ischemia (Non-Patent Document 1).

Moreover, it is known that activins exert an anti-inflammatory effect byinducing M2 macrophage (Patent Document 2), and that animmunosuppressive agent containing adipose tissue-derived mesenchymalstem cells induces M2 macrophage (Patent Document 3).

However, it has not been disclosed that vitamin B₁₂ promotes M2macrophage/microglia induction, inhibits M1 macrophage/microgliainduction, and alleviates neurological diseases such as cerebralinfarction.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: Japanese Patent Laid-Open No. 2016-513694-   Patent Document 2: International Publication No. WO 2011/149036-   Patent Document 3: International Publication No. WO 2011/043136

Non-Patent Document

-   Non-Patent Document 1: Current Medicinal Chemistry, 2007, Vol. 14,    No. 3, p. 249-263

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

An object of the present invention is to provide a therapeutic agent fornervous system disease. Preferably, an object of the present inventionis to provide a therapeutic agent for nervous system disease having atleast one selected from the group consisting of an apoptosis inhibitingeffect, a necrosis inhibiting effect, an axon outgrowth promotingeffect, an M2 macrophage/microglia induction promoting effect, an M1macrophage/microglia induction inhibiting effect, and a nerveregeneration promoting effect.

Means for Solving the Problems

The present inventors have made extensive investigations in view of theabove problems and have found that vitamin B₁₂ has a therapeutic effecton nervous system diseases. In addition, the present inventors also havefound that vitamin B₁₂ has an apoptosis inhibiting effect, a necrosisinhibiting effect, an axon outgrowth promoting effect, an M2macrophage/microglia induction promoting effect, an M1macrophage/microglia induction inhibiting effect, a nerve regenerationpromoting effect, and the like. The present inventors have made furtherinvestigations based on these findings and have accomplished the presentinvention.

Specifically, the present invention encompasses the following aspects:

Item 1. A therapeutic agent for nervous system disease comprisingvitamin B₁₂.

Item 1A. A method of treating a nervous system disease, the methodcomprising administering vitamin B₁₂ to a patient in need of treatmentof a nervous system disease.

Item 1B1. Vitamin B₁₂ for use in treating a nervous system disease.

Item 1B2. A composition comprising vitamin B₁₂ for use in treating anervous system disease.

Item 1C. Use of vitamin B₁₂ for the manufacture of a therapeutic agentfor nervous system disease.

Item 2. The therapeutic agent for nervous system disease according toItem 1, wherein the therapeutic agent for nervous system disease is anM2 macrophage/microglia induction promoting agent.

Item 3. The therapeutic agent for nervous system disease according toItem 1, wherein the therapeutic agent for nervous system disease is anM1 macrophage/microglia induction inhibiting agent.

Item 4. The therapeutic agent for nervous system disease according toany one of Items 1 to 3, wherein the therapeutic agent for nervoussystem disease is a nerve regeneration promoting agent.

Item 5. The therapeutic agent for nervous system disease according toany one of Items 1 to 4, wherein the nervous system disease is a centralnervous system disease.

Item 6. The therapeutic agent for nervous system disease according toItem 5, wherein the central nervous system disease is a cerebrovasculardisease.

Item 7. The therapeutic agent for nervous system disease according toItem 6, wherein the cerebrovascular disease is at least one selectedfrom the group consisting of cerebral infarction, cerebral hemorrhage,cerebral thrombosis, cerebral arteriosclerosis, and dementia.

Item 8. The therapeutic agent for nervous system disease according toany one of Items 1 to 4, wherein the nervous system disease is nerveinjury.

Item 9. The therapeutic agent for nervous system disease according toItem 8, wherein the nerve injury is central nerve injury.

Item 10. The therapeutic agent for nervous system disease according toItem 9, wherein the central nerve injury is spinal cord injury.

Item 11. The therapeutic agent for nervous system disease according toany one of Items 1 to 10, wherein the vitamin B₁₂ is at least oneselected from the group consisting of methylcobalamin, cyanocobalamin,hydroxocobalamin, sulfitocobalamin, adenosylcobalamin, and saltsthereof.

Item 12. The therapeutic agent for nervous system disease according toany one of Items 1 to 11, wherein the vitamin B₁₂ is methylcobalamin.

Item 13. The therapeutic agent for nervous system disease according toany one of Items 1 to 12, wherein the therapeutic agent for nervoussystem disease is used for continuous administration.

Item 14. The therapeutic agent for nervous system disease according toItem 13, wherein the therapeutic agent for nervous system disease is aformulation for intravenous drip infusion.

Item 15. The therapeutic agent for nervous system disease according toany one of Items 1 to 14, wherein the therapeutic agent for nervoussystem disease is used so that administration is started at 12 to 24hours after onset of the disease.

Item 16. The therapeutic agent for nervous system disease according toany one of Items 1 to 14, wherein the therapeutic agent for nervoussystem disease is used so that administration is started immediatelyafter onset of the disease or within 12 hours of the onset.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of TUNEL assay in Example 1. The vertical axisindicates the proportion of apoptotic cells. The horizontal axisindicates types of drugs added and whether the drugs were added (+) ornot (−). Two asterisks “**” indicates that the result of a statisticalanalysis using the Tukey-Kramer method had a p value of less than 0.01.

FIG. 2 shows the results of lactate dehydrogenase (LDH) assay in Example2. The vertical axis indicates LDH activity, which is an index ofnecrosis, as a percentage with respect to that in a high control. Thehorizontal axis indicates a type of a drug added and whether the drugwas added (10 μM) or not (−). An asterisk “*” indicates that the resultof a statistical analysis using Student's t-test had a p value of lessthan 0.05.

FIG. 3 shows the results of a neurite outgrowth assay in Example 3. Thevertical axis indicates the average length of 30 or more neural axons.The horizontal axis indicates concentrations of a drug added. Thecontrol (CTR) contains no drug. An asterisk “*” indicates that theresult of a statistical analysis using Dunnett's test against CTR had ap value of less than 0.05, and “**” indicates that the result had a pvalue of less than 0.01.

FIG. 4 shows the results of 2,3,5-triphenyltetrazolium chloride (TTC)staining in Example 4. The vertical axis indicates the ischemic lesionvolume. The horizontal axis indicates a type of a drug added and whetherthe drug was added (MeCb1) or not (Control). Two asterisks “**”indicates that the result of a statistical analysis using Student'st-test had a p value of less than 0.01.

FIG. 5 shows the results of Western blot in Example 5. The vertical axesindicate the ratio of the amount of an M1 marker (left graph, IL- 1βprotein; right graph, iNOS protein) to the amount ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. The horizontalaxes indicate types of drugs added and whether the drugs were added (+or concentration) or not (−). An asterisk “*” indicates that the resultof a statistical analysis using Dunnett's test had a p value of lessthan 0.05, and “**” indicates that the result had a p value of less than0.01.

FIG. 6 shows the results of Western blot in Example 5. The vertical axesindicate the ratio of the amount of an M2 marker (left graph, arginase I(Arg1) protein; right graph, CD206 protein) to the amount of GAPDHprotein. The horizontal axes indicate types of drugs added and whetherthe drugs were added (+ or concentration) or not (−). An asterisk “*”indicates that the result of a statistical analysis using Dunnett's testhad a p value of less than 0.05, and “**” indicates that the result hada p value of less than 0.01.

FIG. 7 shows the results of an immunohistological evaluation in Example5. The vertical axes indicate the percentage of M1 macrophages(percentage of iNOS-positive cells) in the left graph and the percentageof M2 macrophages (percentage of Arg1-positive cells) in the rightgraph. The horizontal axes indicate types of drugs added and whether thedrugs were added (+ or concentration) or not (−). Two asterisks “**”indicates that the result of a statistical analysis using Dunnett's testhad a p value of less than 0.01.

FIG. 8-1 shows the results of Western blot in Example 6. The verticalaxis indicates the ratio of the amount of phosphorylated Akt protein tothe amount of Akt protein. The horizontal axes indicate types of drugsadded and whether the drugs were added (+ or concentration) or not (−).An asterisk “*” indicates that the result of a statistical analysisusing the Tukey-Kramer method had a p value of less than 0.05, “**”indicates that the result had a p value of less than 0.01, and “***”indicates that the result had a p value of less than 0.001.

FIG. 8-2 shows the results of Western blot in Example 6. The verticalaxis indicates the ratio of the amount of phosphorylated 4EBP1 proteinto the amount of 4EBP1 protein. The horizontal axes indicate types ofdrugs added and whether the drugs were added (+ or concentration) or not(−). An asterisk “*” indicates that the result of a statistical analysisusing the Tukey-Kramer method had a p value of less than 0.05, “**”indicates that the result had a p value of less than 0.01, and “***”indicates that the result had a p value of less than 0.001.

FIG. 8-3 shows the results of Western blot in Example 6. The verticalaxis indicates the ratio of the amount of phosphorylated S6K protein tothe amount of S6K protein. The horizontal axes indicate types of drugsadded and whether the drugs were added (+ or concentration) or not (−).An asterisk “*” indicates that the result of a statistical analysisusing the Tukey-Kramer method had a p value of less than 0.05, “**”indicates that the result had a p value of less than 0.01, and “***”indicates that the result had a p value of less than 0.001.

FIG. 9 shows the results of immunohistological evaluation in Example 7.Graphs, from the left row, show the results at 2.5 mm in the proximaldirection from an injury site, the injury site, and 2.5, 5.0, and 7.5 mmin the distal direction. The vertical axes indicate the number ofmacrophages in the upper graphs, the number of M1 macrophages in themiddle graphs, and the proportion of M1 macrophages in the lower graphs.The horizontal axes indicate the number of days elapsed after occurrenceof sciatic nerve injury. CTR refers to an untreated group, MeCb1 refersto a methylcobalamin treatment group, and Sham refers to a non-injurygroup, which underwent only sciatic nerve exteriorization. An asterisk“*” indicates that the result of a statistical analysis using theTukey-Kramer method (CTR vs. MeCbl) had a p value of less than 0.05, and“#” indicates that the result of a statistical analysis using Student'st-test (CTR vs. MeCbl) had a p value of less than 0.05.

FIG. 10 shows the results of immunohistological evaluation in Example 7.Graphs, from the left row, show the results at 2.5 mm in the proximaldirection from an injury site, the injury site, and 2.5, 5.0, and 7.5 mmin the distal direction. The vertical axes indicate the number ofmacrophages in the upper graphs, the number of M2 macrophages in themiddle graphs, and the proportion of M2 macrophages in the lower graphs.The horizontal axes indicate the number of days elapsed after occurrenceof sciatic nerve injury. CTR refers to an untreated group, MeCbl refersto a methylcobalamin treatment group, and Sham refers to a non-injurygroup, which underwent only sciatic nerve exteriorization. An asterisk“*” indicates that the result of a statistical analysis using theTukey-Kramer method (CTR vs. MeCbl) had a p value of less than 0.05, and“#” indicates that the result of a statistical analysis using Student'st-test (CTR vs. MeCbl) had a p value of less than 0.05.

FIG. 11 shows the results of an immunohistological evaluation in Example8. The vertical axes indicate the number of axons in the upper graph,the number of myelinated axons in the middle graph, and the myelinationratio in the lower graph. The horizontal axes represent positions atwhich transverse sections of a nerve were prepared (from left, 2.5 mm inthe proximal direction from an injury site, the injury site, and 2.5,5.0, and 7.5 mm in the distal direction). CTR refers to an untreatedgroup, MeCbl refers to a methylcobalamin treatment group, and Shamrefers to a non-injury group, which underwent only sciatic nerveexteriorization. An asterisk “*” indicates that the result of astatistical analysis using the Tukey-Kramer method (CTR vs. MeCbl) had ap value of less than 0.05.

FIG. 12 shows the results of measurement of BBB scores in Example 9. Thevertical axis indicates the BBB score. The horizontal axis indicates thenumber of days elapsed after a surgery for constructing a spinal cordinjury model, with 0 indicating pre-surgery. CTR refers to an untreatedgroup, MeCbl refers to a methylcobalamin treatment group, and Shamrefers to a non-injury group, which underwent only sciatic nerveexteriorization. An asterisk “*” indicates that the result of astatistical analysis using Steel-Dwass test (CTR vs. MeCbl) had a pvalue of less than 0.05.

FIG. 13 shows the results of thermal algesimetry test in Example 9. Thevertical axis indicates the time after applying infrared thermalstimulation to the right sole of the hind limb of a model rat until therat retracted the limb because of the applied heat. The horizontal axisindicates the number of days elapsed after a surgery for constructing aspinal cord injury model, with 0 indicating pre-surgery. CTR refers toan untreated group, MeCbl refers to a methylcobalamin treatment group,and Sham refers to a non-injury group, which underwent only sciaticnerve exteriorization. An asterisk “*” indicates that the result of astatistical analysis using Steel-Dwass test (CTR vs. MeCbl) had a pvalue of less than 0.05.

FIG. 14 shows the results of Western blot of M1 markers (IL-1β proteinand iNOS protein) in Example 10. The vertical axes indicate the ratio ofthe amount of the M1 marker (IL-1β protein or iNOS protein) to theamount of GAPDH protein. The horizontal axes indicate types of drugsadded and whether the drugs were added (+ or concentration) or not (−).An asterisk “*” indicates that the result of a statistical analysisusing Dunnett's test had a p value of less than 0.05, and “**” indicatesthat the result had a p value of less than 0.01.

FIG. 15 shows the results of Western blot of M2 markers (Arg1 proteinand CD206 protein) in Example 10. The vertical axes, the horizontalaxes, and each symbol have the same meaning as described for FIG. 14 .

FIG. 16 shows the results of immunofluorescent staining at 7 days afterthe surgery in Example 11. Each graph shows difference depending on theposition. The horizontal axis in each graph indicates the direction anddistance from an injury site. The vertical axes indicate the number ofmacrophages in the upper graphs, the number of M1 or M2 macrophages inthe middle graphs, and the proportion of M1 or M2 macrophages in thelower graph. CTR refers to an untreated group, and MeCbl refers to amethylcobalamin treatment group. An asterisk “*” indicates that theresult of a statistical analysis using Mann Whitney U test (CTR vs.MeCbl) had a p value of less than 0.05, and “**” indicates that theresult had a p value of less than 0.01.

FIG. 17 shows the results of immunofluorescent staining at 14 days afterthe surgery in Example 11 and is otherwise the same as FIG. 16 .

FIG. 18 shows the results of immunofluorescent staining at 28 days afterthe surgery in Example 11 and is otherwise the same as FIG. 16 .

FIG. 19 shows the results of immunofluorescent staining of M1 macrophagein Example 11. Each graph shows difference depending on the number ofdays elapsed after the surgery. Graphs in each row show the resultsaccording to the direction and distance from an injury site: 2 and 1 mmon the head side and 1 and 2 mm on the tail side. The vertical axesindicate the number of macrophages in the upper graphs, the number of M1macrophages in the middle graphs, and the proportion of M1 macrophagesin the lower graph. The horizontal axes indicate the number of dayselapsed after the surgery for constructing a spinal cord injury model.CTR refers to an untreated group, and MeCbl refers to a methylcobalamintreatment group. An asterisk “*” indicates that the result of astatistical analysis using Mann Whitney U test (CTR vs. MeCbl) had a pvalue of less than 0.05, and “**” indicates that the result had ap valueof less than 0.01.

FIG. 20 shows the results of immunofluorescent staining of M2 macrophagein Example 11 and is otherwise the same as FIG. 19 .

FIG. 21 shows the results of immunofluorescent staining for the M1/M2ratio in Example 11. Each graph shows difference depending on the numberof days elapsed after the surgery. The graphs show the results accordingto the direction and distance from the injury site: 2 and 1 mm on thehead side and 1 and 2 mm on the tail side. The vertical axes indicatethe M1/M2 ratio. The horizontal axes indicate the number of days elapsedafter the surgery for constructing a spinal cord injury model. CTRrefers to an untreated group, and MeCbl refers to a methylcobalamintreatment group. An asterisk “*” indicates that the result of astatistical analysis using Mann Whitney U test (CTR vs. MeCbl) had a pvalue of less than 0.05, and “**” indicates that the result had a pvalue of less than 0.01.

FIG. 22 shows the results of immunofluorescent staining for the M1/M2ratio in Example 11. Each graph shows difference depending on theposition. The graphs, from left, show the results at 7, 14, and 28 daysafter a surgery for constructing a spinal cord injury model. Thevertical axes indicate the M1/M2 ratio. The horizontal axes indicate thenumber of days elapsed after the surgery for constructing a spinal cordinjury model. The horizontal axes indicate the direction and distancefrom the injury site. CTR refers to an untreated group, and MeCbl refersto a methylcobalamin treatment group. An asterisk “*” indicates that theresult of a statistical analysis using Mann Whitney U test (CTR vs.MeCbl) had a p value of less than 0.05, and “**” indicates that theresult had a p value of less than 0.01.

FIG. 23 shows the results of a rotarod test in Example 12. Thehorizontal axis indicates the number of days elapsed after a surgerycreating cerebral infarction, and the vertical axis indicates relativetime until a model mouse fell from a rotarod. CTR refers to an untreatedgroup, and MeCbl refers to a methylcobalamin treatment group. Twoasterisks “**” indicates that the result of a statistical analysis usingMann Whitney U test (CTR vs. MeCbl) had a p value of less than 0.01.

MODE FOR CARRYING OUT THE INVENTION

As used herein, the expressions “containing” and “comprising” encompassconcepts of “containing,” “comprising,” “substantially comprising,” and“consisting of.”

As used herein, the term “macrophage/microglia” refers to “macrophagesand/or microglia” and encompasses both the meaning of “macrophages andmicroglia” and the meaning of “macrophages or microglia.”

In one aspect, the present invention relates to a therapeutic agent fornervous system disease, an apoptosis inhibiting agent, a necrosisinhibiting agent, an axon outgrowth promoting agent, an M2macrophage/microglia induction promoting agent, an M1macrophage/microglia induction inhibiting agent, a nerve regenerationpromoting agent, or the like that contains vitamin B₁₂ (herein, may alsobe referred to as an “agent of the present invention”). These agentswill be described below.

1. Active Ingredient

Vitamin B₁₂ includes cobalamin, derivatives thereof, and salts of theforegoing. Specific examples of vitamin B₁₂ include cobalamin, a cobaltsubstitution product of cobalamin, and derivatives thereof. Morespecific examples include methylcobalamin, cyanocobalamin,hydroxocobalamin, sulfitocobalamin, adenosylcobalamin, and saltsthereof. Among these, methylcobalamin, cyanocobalamin, hydroxocobalamin,and salts thereof are preferred, and methylcobalamin and salts thereofare more preferred.

The salts of cobalamin and derivatives thereof are not particularlylimited as long as the salts are pharmacologically acceptable, and bothacid salts and basic salts can be used. Examples of the acid saltinclude inorganic acid salts such as hydrochlorides, hydrobromides,sulfates, nitrates, and phosphates; organic acid salts such as acetates,propionates, tartrates, fumarates, maleates, malates, citrates,methanesulfonates, and p-toluenesulfonates; and amino acid salts such asaspartates and glutamates. Examples of the basic salt include alkalimetal salts such as sodium salts and potassium salts; and alkaline-earthmetal salts such as calcium salts and magnesium salts.

Vitamin B₁₂ may be in the form of solvates. The solvents are notparticularly limited as long as the solvents are pharmacologicallyacceptable, and examples include water, ethanol, glycerol, and aceticacid.

As Vitamin B₁₂, one type may be used alone, or two or more type may beused in combination.

2. Use

Vitamin B₁₂ has a therapeutic effect on a nervous system disease.Therefore, vitamin B₁₂ can be used as an active ingredient in atherapeutic agent for nervous system disease.

Vitamin B₁₂ has an apoptosis inhibiting effect, a necrosis inhibitingeffect, an axon outgrowth promoting effect, an M2 macrophage/microgliainduction promoting effect, an M1 macrophage/microglia inductioninhibiting effect, a nerve regeneration promoting effect, and the like.Therefore, vitamin B₁₂ can be used as an active ingredient of agentssuch as an apoptosis inhibiting agent, a necrosis inhibiting agent, anaxon outgrowth promoting agent, an M2 macrophage/microglia inductionpromoting agent, an M1 macrophage/microglia induction inhibiting agent,an M1:M2 ratio (ratio of M1 macrophage/microglia to M2macrophage/microglia) reducing agent, or a nerve regeneration promotingagent.

In addition, vitamin B₁₂ may be used as an active ingredient in apreferred form of the therapeutic agent for nervous system disease,which is an active ingredient in a therapeutic agent for nervous systemdisease based on at least one selected from the group consisting of anapoptosis inhibiting effect, a necrosis inhibiting effect, an axonoutgrowth promoting effect, an M2 macrophage/microglia inductionpromoting effect, an M1 macrophage/microglia induction inhibitingeffect, and a nerve regeneration promoting effect.

The nervous system disease is not particularly limited and includes acentral nervous system disease and a peripheral nervous system disease.Examples of the central nervous system disease include a cerebrovasculardisease and central nervous system injury.

Examples of the cerebrovascular disease include cerebral infarction,cerebral hemorrhage, cerebral thrombosis, cerebral arteriosclerosis, anddementia.

The nerve injury may be either peripheral nerve injury or central nerveinjury. The central nerve injury includes spinal cord injury. Causes ofthe nerve injury are not particularly limited, and nerve injuries of avariety of causes, such as traumatic injury, pressure caused by aplaster cast, electrical injury, disk herniation, or radiation exposuremay be applicable. The severity of applicable nerve injuries is notparticularly limited, and applicable cases include all of casesincluding a case where axons are preserved but demyelination hasoccurred, a case where Wallerian degeneration is accompanied, and a casewhere nerves are anatomically divided. The nerve injury encompassesvarious symptoms associated with the nerve injury including, forexample, dyskinesia (e.g., motor paralysis and muscle weakness in upperand lower extremities), sensory disorder (e.g., hypesthesia, numbness,and pain), autonomic nerve disorder (e.g., dyshidrosis, change of skincolor), or the like in an injured innervation area.

The nervous system disease is preferably a nervous system disease thatcan be treated by at least one selected from the group consisting of anapoptosis inhibiting effect, a necrosis inhibiting effect, an axonoutgrowth promoting effect, an M2 macrophage/microglia inductionpromoting effect, an M1 macrophage/microglia induction inhibitingeffect, and a nerve regeneration promoting effect.

An agent of the present invention is not particularly limited as long asthe agent contains vitamin B₁₂ (as used herein, may be referred tosimply as an “active ingredient”) and may contain other ingredients, ifnecessary. The other ingredients are not limited as long as theingredients are pharmacologically acceptable. The other ingredientsinclude additives in addition to an ingredient having a pharmacologicalaction. Examples of the additives include bases, carriers, solvents,dispersants, emulsifiers, buffers, stabilizers, excipients, binders,disintegrants, lubricants, thickeners, humectants, colorants, flavoringagents, and chelating agents.

Vitamin B₁₂ used alone can exert a nervous system disease therapeuticeffect, an apoptosis inhibiting effect, a necrosis inhibiting effect, anaxon outgrowth promoting effect, an M2 macrophage/microglia inductionpromoting effect, an M1 macrophage/microglia induction inhibiting effect(here, an effect of switching M1 macrophage/microglia to M2macrophage/microglia is not ruled out), a nerve regeneration promotingeffect, and the like. Therefore, the agent of the present invention canexert the desired effect without containing other ingredients havingthese effects and/or actions. However, the agent of the presentinvention can contain other ingredients having a pharmacological action.

Modes of use for the agent of the present invention are not particularlylimited, and a suitable mode of use can be selected according to thetype of the agent. The agent of the present invention can be used invitro (e.g., added to a medium of cultured cells) or in vivo (e.g.,administered to animals), for example, depending on the purpose.

Targets for application of the agent of the present invention are notparticularly limited. Examples of target mammals include humans,monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep,goats, and deer. Examples of target cells include animal cells. Types ofcells are not particularly limited either, and examples include bloodcells, hematopoietic stem cells, progenitor cells, gametes (sperm andovum), fibroblasts, epithelial cells, vascular endothelial cells, nervecells, hepatic cells, keratinocytes, muscle cells, epidermal cells,endocrine cells, ES cells, iPS cells, tissue stem cells, and cancercells.

The agent of the present invention can be in any dosage form. Examplesof the dosage form include oral dosage forms such as tablets (includingorally disintegrating tablets, chewable tablets, foam tablets, lozenges,and gelatinous drop formulations), pills, granules, fine granules,powders, hard capsules, soft capsules, dry syrup preparations, liquids(including drinkable formulations, suspensions, and syrups), and jellyformulations; and parenteral dosage forms such as injection formulations(e.g., drip infusions [e.g., formulations for intravenous dripinfusion], intravenous injections, intramuscular injections,subcutaneous injections, and intradermal injections), topical agents(e.g., ointments, plasters, and lotions), suppositories, inhalants,ophthalmic formulations, ophthalmic ointments, nasal drops, ear drops,and liposome formulations.

Administration routes of the agent of the present invention are notparticularly limited as long as a desired effect can be achieved, andexamples include oral administration and parenteral administrationincluding enteral administration, such as tube feeding and enemaadministration, intravenous administration, intraarterialadministration, intramuscular administration, intracardiacadministration, subcutaneous administration, intradermal administration,and intraperitoneal administration.

The content of an active ingredient in the agent of the presentinvention is not limited and varies depending on the mode of use, theapplication target, conditions of the application target, and the like.For example, the content can be 0.0001 to 100 wt % and preferably 0.001to 50 wt %.

The dose of the agent of the present invention for administration toanimals is not particularly limited as long as the dose is an effectivedose, which produces medicinal benefit. The usual dose (weight of anactive ingredient) is 0.1 to 1000 mg/kg body weight per day andpreferably 0.5 to 500 mg/kg body weight per day for oral administration;and 0.01 to 100 mg/kg body weight per day and preferably 0.05 to 50mg/kg body weight per day for parenteral administration. Theabove-described doses may be suitably adjusted depending on the age,pathological conditions, and symptoms.

The agent of the present invention is preferably used by continuousadministration from the viewpoint of further effectively achieving theM2 macrophage/microglia induction promoting effect, the M1macrophage/microglia induction inhibiting effect, the nerve regenerationpromoting effect, or the like. By continuous administration, theconcentration of an active ingredient, which acts on cells in the targetof administration (e.g., cells in the affected area of a nervous systemdisease, preferably macrophage/microglia), can be maintained withinconcentrations suitable for achieving the M2 macrophage/microgliainduction promoting effect, the M1 macrophage/microglia inductioninhibiting effect, and the nerve regeneration promoting effect (e.g., 5to 100 μM, preferably 10 to 50 μM, more preferably 20 to 10 μM, stillmore preferably 50 to 5 μM, and even more preferably 100 to 1 μM), andthe effects can be more effectively achieved. In addition, the agent ofthe present invention is preferably a drip infusion, and more preferablya formulation for intravenous drip infusion, although depending on thetarget of application.

Timing for administration of the agent of the present invention is notparticularly limited.

As an example, the agent of the present invention is used so thatadministration of the agent is started at 12 to 24 hours after onset ofthe disease. The onset is a time when a symptom of a disease or a factordirectly causing the disease can be recognized. For example, the onsetis a time at which an ischemic lesion develops in an ischemiccerebrovascular disease such as cerebral infarction. Since the agent ofthe present invention can act on the repair mechanism, which can operaterelatively long after the development of the ischemic lesion (an M2macrophage/microglia induction promoting effect, an M1macrophage/microglia induction inhibiting effect, and a nerveregeneration promoting effect), therapeutic effects can be achieved evenif the agent is administered at the above-described timing (at 12 to 24hours after onset) in the application of the agent to an ischemiccerebrovascular disease such as cerebral infarction.

As another example, the agent of the present invention is used so thatadministration of the agent is started during an acute phase (e.g.,immediately after onset or within 12 hours after onset) of a nervoussystem disease such as nerve injury (preferably spinal cord injury). Theonset is a time when a symptom of a disease or a factor directly causingthe disease can be recognized. For example, in nerve injury such asspinal cord injury, the onset is a time at which a nerve is injured.

EXAMPLES

The present invention will be described in detail below based onexamples, but the present invention is not limited by the examples.

Example 1. Apoptosis Inhibiting Effect on Cerebral Cortical Neurons

The apoptosis inhibiting effect of methylcobalamin on cerebral corticalneurons was examined by TUNEL assay. Specifically, the examination wascarried out as follows.

<Example 1-1. Preparation of Cerebral Cortical Neurons>

Cerebral cortical neurons were collected and cultured by a conventionalmethod. Cerebral cortex was dissected from a fetus of a Sprague-Dawley(SD) rat (18th day of pregnancy) and recovered in ice-cooled Dulbecco'sModified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and1% penicillin/streptomycin. The pia mater and blood vessels wereremoved, and the remaining cerebral cortex was transferred to DMEM(containing 1% penicillin/streptomycin and no FBS) and cut into smallpieces having a size of 1 mm by surgical scissors. Papain (finalconcentration, 2 mg/ml) was added to the cell mixture, and the mixturewas allowed to react at 37° C. for 30 minutes. DNase I (70 U/ml) wasadded to the reaction mixture and reaction was carried out for 30seconds. Then, DMEM containing 10% FBS and 1% penicillin/streptomycinwas added to terminate the reaction. The cell mixture was centrifuged at800 rpm, the residue was re-suspended in DMEM containing 10% FBS and 1%penicillin/streptomycin, and the suspension was seeded on a dish coatedwith poly-L lysine (PLL). At 4 hours after the cell seeding, the mediumwas replaced with Neurobasal medium (containing 10% B27 supplement and1% penicillin/streptomycin).

<Example 1-2. TUNEL Assay>

To the cerebral cortical neurons (Example 1-1) cultured on a PLL-coated8-well chamber slide, 20 mM glutamic acid and 10 μM methylcobalamin(MeCbl) were added. After 18 hours, the proportion of apoptotic cellswas evaluated using Promega's DeadEnd Fluorometric TUNEL System. Cellswere fixed with 4% paraformaldehyde (PFA) at 4° C. for 25 minutes. Aftercells were permeabilized with 0.2% Triton X-100 for 5 minutes, anincubation buffer was added, and the mixture was left stand at 37° C.with light shielding for 60 minutes to label the permeabilized cells.The nuclei were labeled with 4′6-diamidino-2-phenylindole (DAPI). Thetotal cell number and the number of TUNEL-positive cells weredetermined.

<Example 1-3. Results>

The results are shown in FIG. 1 . The values corresponding to data inthe graph of FIG. 1 are shown in Table 1. In the TUNEL assay, whenmethylcobalamin was added alone, the proportion of apoptotic cells (%)was similar to that in the control. When glutamic acid was added alone,the proportion of apoptotic cells was significantly increased. However,when methylcobalamin was added together with glutamic acid, theproportion of apoptotic cells was significantly decreased to the levelof the control.

TABLE 1 Glutamate − − + + MeCbl − + − + Average 6.0 5.9 15.0 5.6Standard error 1.2 1.3 1.3 0.6 Standard deviation 2.0 2.2 2.2 1.1

Example 2. Necrosis Inhibiting Effect on Cerebral Cortical Neurons

A necrosis inhibiting effect of methylcobalamin on cerebral corticalneurons was examined by LDH assay. Specifically, the examination wascarried out as follows.

<Example 2-1. LDH Assay>

To the cerebral cortical neurons (Example 1-1) cultured on a PLL-coated6-well chamber slide, 10 μM methylcobalamin was added at 30 minutesbefore exposure to oxygen-glucose deprivation (OGD) stress.N-methyl-D-aspartic acid (NMDA) was added to a high control as areference. The medium was replaced with Earle's balanced salt solution(EBSS), and the neurons were exposed to the OGD stress in an atmospherewith an oxygen concentration of 1% for 3 hours. The neurons werereturned to a normal medium and an atmosphere with a normal oxygenconcentration. After 24 hours, the supernatant was collected, and LDHactivity was measured using Cytotoxicity Detection Kit^(PLUS) (SIGMA).LDH activities of the control group and the methylcobalamin additiongroup were calculated as proportions (%) with respect to the LDHactivity of the high control.

<Example 2-2. Results>

The results are shown in FIG. 2 . The values corresponding to data inthe graph of FIG. 2 are shown in Table 2. In the LDH assay under OGDstress, the proportion of LDH activity in the methylcobalamin additiongroup with respect to the high control was significantly decreased ascompared with that in the control group.

TABLE 2 MeCbl (−) MeCbl (+) Average 3.1 1.3 Standard error 0.6 0.5Standard deviation 1.0 0.8

Example 3. Axon Outgrowth Promoting Effect on Cerebral Cortical Neurons

An axon outgrowth promoting effect of methylcobalamin on cerebralcortical neurons was examined by a neurite outgrowth assay.Specifically, the examination was carried out as follows.

<Example 3-1. Neurite Extension Assay>

The cerebral cortical neurons (Example 1-1) were seeded, and the drugwas added in different concentrations at 24 hours. The concentrations ofmethylcobalamin added were 1, 10, and 100 nM and 1, 10, and 100 μM. At72 hours after the cell seeding, immunofluorescent staining wasperformed using an anti-TuJ1 antibody, and the lengths of axons (thelongest neurite length per neuron) were measured. The measurement wasperformed on cells that were not in contact with other cells. In eachevaluation, at least 30 neural axons were measured, and the mean of theobtained values was calculated and designated as a measured value.

<Example 3-2. Results>

The results are shown in FIG. 3 . The values corresponding to data inthe graph of FIG. 3 are shown in Table 3. The results of the neuriteoutgrowth assay showed trends for promotion of axon outgrowth dependenton the concentration, with a peak at a methylcobalamin concentration of10 μM. At 1 and 10 μM concentrations, the axon outgrowth wassignificantly promoted as compared with the control group containing nodrug.

TABLE 3 CTR 1 nM 10 nM 100 nM 1 μM 10 μM 100 μM Average 64.8 67.2 71.275.2 79.9 86.4 66.2 Standard 2.9 3.1 2.3 2.8 1.6 4.1 2.2 error Standard6.6 7.0 5.2 6.4 3.5 9.1 4.9 deviation

Example 4. Brain Ischemic Lesion Volume Reducing Effect

A brain ischemic lesion volume reducing effect of methylcobalamin wasexamined using a 2,3,5-triphenyltetrazolium chloride (TTC) stainingmethod. Specifically, the examination was carried out as follows.

<Example 4-1. Construction of Transient Middle Cerebral Artery Occlusion(tMCAO) Model and Administration of Drug>

Eight- to 9-week-old male C57BL/6J mice (about 24 g) were used. A probefor a laser Doppler blood flow meter was placed on the right skull suchthat blood flow in the middle cerebral artery could be monitored. Theright cervical region was cut and opened, and the external carotidartery was ligated. Then, the common carotid artery was cut, and a nylonthread was inserted into the common carotid artery, and the tip wasadvanced while the blood flow was checked using a blood flow monitor.When the tip reached the bifurcation of the middle cerebral artery and adecrease in blood flow was observed, this state was held for 1 hour at arectal temperature of 37° C. Then, the nylon thread was removed, and thecommon carotid artery was ligated. In order to administermethylcobalamin continuously, an osmotic minipump was placed and left inthe dorsal subcutaneous space. After the model had been constructed,methylcobalamin was administered at a dose of 1 mg/kg/day. In anuntreated group, physiological saline was administered instead ofmethylcobalamin according to the same procedure. After the surgery, thetemperature was maintained with a rectal temperature of 37° C. until themouse recovered from anesthesia.

<Example 4-2. TTC Staining Method>

At 2 days after the surgery, the mouse (Example 4-1) was sacrificed, andthe cerebrum was excised. The cerebrum was cut at intervals of 1 mm toprepare coronal sections, and the sectioned slices were immersed in 2%TTC solution for 30 minutes. Images were obtained by a stereoscopicmicroscope, the ischemic lesion area in each slice was calculated, andall ischemic lesion areas of the cerebrum slices were summed up tocalculate an ischemic lesion volume. The ischemic lesion area in eachslice was calculated by the following formula: Area of unaffectedhemisphere—area of affected side intact portion.

<Example 4-3. Results>

The results are shown in FIG. 4 . The values corresponding to data inthe graph of FIG. 4 are shown in Table 4. At 2 days after the tMCAOsurgery, the brain ischemic lesion volume was evaluated using TTCstaining. In the methylcobalamin treatment group, the ischemic lesionvolume was significantly reduced by about ½ as compared with the controlgroup.

TABLE 4 Control MeCbl Average 59.1 28.8 Standard error 8.0 3.2 Standarddeviation 22.6 9.0

Example 5. M2 Macrophage Induction Promoting Effect and M1 MacrophageInduction Inhibiting Effect

An M2 macrophage induction promoting effect and an M1 macrophageinduction inhibiting effect of methylcobalamin were examined by aWestern blot method and an immunohistological evaluation method.Specifically, the examination was carried out as follows.

<Example 5-1. Preparation of Macrophage Cell Line>

Mouse macrophage cell line J774A.1 (JCRB9108) was purchased from JCRBCell Bank (Laboratory of Cell Cultures), Osaka, Japan. Cells werecultured in DMEM containing 10% FBS and 1% penicillin/streptomycin.

<Example 5-2. Western Blot>

The J774A.1 cells (Example 5-1) were seeded on a dish having a diameterof 6 cm. After 4 days, proteins were collected using a cell lysis buffercontaining a cocktail of protease inhibitors. The protein concentrationwas measured by BCA assay. Then, 50 μg of each sample was subjected toSDS-PAGE, and the electrophoresed proteins were transferred to apolyvinylidene difluoride membrane. The membrane was blocked with ablocking buffer for 1 hour, and then the proteins were allowed to reactwith primary antibodies at 4° C. overnight. The proteins were furtherallowed to react with a secondary antibody at room temperature for 1hour and detected using ECL Western Blotting Detection System. To detectthe M1 markers iNOS and IL-1β, lipopolysaccharide (LPS) (100 ng/ml) andmethylcobalamin were added at 24 hours before collecting the proteins.To detect the M2 markers Arg1 and CD206, IL-4 (20 ng/ml) andmethylcobalamin were added at 72 hours before collecting the proteins.

The primary antibodies used were a rabbit anti-IL-1(3 polyclonalantibody (Santa Cruz), a rabbit anti-iNOS monoclonal antibody (Abcam), arabbit anti-Arg1 polyclonal antibody (Santa Cruz), and a rabbitanti-CD206 monoclonal antibody (Abcam). The secondary antibody used wasanti-rabbit IgG, horseradish peroxidase-linked whole antibody fromdonkey (GE Healthcare Life Sciences).

<Example 5-3. Method for Immunohistological Evaluation>

The J774A.1 cells (Example 5-1) were seeded on a dish having a diameterof 6 cm. After 4 days, cells were fixed with 4% PFA for 20 minutes.Blocking was performed for 30 minutes, and the cells were allowed toreact with primary antibodies at 4° C. overnight. The cells were furtherallowed to react with a secondary antibody at room temperature for 1hour so that the nuclei were labeled with DAPI. To detect the M1 markeriNOS, LPS (100 ng/ml) and methylcobalamin were added at 24 hours beforefixing the cells. To detect the M2 marker Arg1, IL-4 (20 ng/ml) andmethylcobalamin were added at 72 hours before fixing the cells.

The primary antibodies used were a rabbit anti-iNOS monoclonal antibody(Abcam) and a rabbit anti-Arg1 polyclonal antibody (Santa Cruz). Thesecondary antibody used was an Alexa 488-labeled goat anti-rabbit IgGantibody (Life Technologies) or an Alexa 568-labeled goat anti-rabbitIgG antibody (Life Technologies).

<Example 5-4. Results>

The results of Western blot are shown in FIGS. 5 and 6 . The valuescorresponding to data in the graphs of FIG. 5 are shown in Table 5, andthe values corresponding to data in the graphs of FIG. 6 are shown inTable 6. The M1 marker levels were as follows: As compared with LPSalone, the IL-1β level was significantly decreased when 100 nMmethylcobalamin was added in combination, and the iNOS level wassignificantly decreased when 100 nM to 10 μM methylcobalamin was addedin combination. As compared with IL-4 alone, the level of the M2 markerArg1 was significantly increased when 100 nM and 1 μM methylcobalaminwas added in combination. Peaks of the CD206 levels were observed whenmethylcobalamin was added at 100 nM to 1 μM.

TABLE 5 LPS − + + + + + + + + MeCbl − − 1 nM 10 nM 100 nM 1 μM 10 μM 100μM 1 mM IL-1β Average 0.01 1.00 0.91 0.68 0.46 0.46 0.60 0.75 1.26Standard error 0.00 0.05 0.14 0.07 0.07 0.19 0.24 0.20 0.04 Standarddeviation 0.00 0.09 0.24 0.11 0.12 0.34 0.41 0.35 0.06 INOS Average 0.011.00 0.87 0.56 0.15 0.27 0.33 0.45 0.53 Standard error 0.00 0.12 0.120.11 0.08 0.04 0.03 0.12 0.38 Standard deviation 0.00 0.21 0.21 0.190.13 0.07 0.04 0.21 0.65

TABLE 6 IL-4 − + + + + + + + + MeCbl − − 1 nM 10 nM 100 nM 1 μM 10 μM100 μM 1 mM Arg1 Average 0.02 1.00 1.42 1.60 1.93 1.65 1.26 0.92 0.54Standard error 0.00 0.19 0.11 0.11 0.20 0.26 0.14 0.17 0.08 Standarddeviation 0.00 0.38 0.21 0.21 0.40 0.53 0.28 0.34 0.17 CD206 Average0.02 1.00 1.28 1.49 1.53 1.58 1.08 1.12 0.78 Standard error 0.00 0.200.31 0.27 0.33 0.17 0.36 0.17 0.63 Standard deviation 0.00 0.39 0.620.55 0.66 0.33 0.73 0.34 1.25

The results of the immunohistological evaluation are shown in FIG. 7 .The values corresponding to data in the graphs of FIG. 7 are shown inTable 7. As compared with LPS alone, the proportion of cells positivefor the M1 marker iNOS was significantly decreased when 10 nM to 100 μMmethylcobalamin was added in combination. As compared to IL-4 addedalone, the proportion of cells positive for the M2 marker Arg1 wassignificantly increased when 10 nM to 1 μM methylcobalamin was added incombination.

In the immunofluorescent staining, a switch from M1 to M2 was observedwith a peak around 100 nM methylcobalamin as in the above-describedWestern blot.

TABLE 7 LPS − + + + + + + + + MeCbl − − 1 nM 10 nM 100 nM 1 μM 10 μM 100μM 1 mM INOS Average 0.00 21.28 16.64 9.57 7.26 3.42 11.45 10.34 17.57Standard error 0.00 1.93 0.63 0.43 1.01 0.70 1.42 2.29 2.22 Standarddeviation 0.00 3.35 1.09 0.75 1.75 1.22 2.47 3.96 3.85 IL-4− + + + + + + + + MeCbl − − 1 nM 10 nM 100 nM 1 μM 10 μM 100 μM 1 mMArg1 Average 1.15 8.22 10.90 18.57 18.95 19.32 15.24 10.61 9.49 Standarderror 0.62 0.26 1.72 1.15 2.48 2.75 2.34 2.68 0.87 Standard deviation1.07 0.45 2.98 2.00 4.29 4.77 4.05 4.64 1.50

Example 6. Analysis of Mechanism of Macrophage Induction Action

The mechanism of the macrophage induction (Example 5) by methylcobalaminwas analyzed. Specifically, the analysis was carried out as follows. At30 minutes after IL-4 and methylcobalamin (100 nM and 1 mM) were added,activation of Akt, 4EBP1, and S6K in the Akt-mTOR pathway (one of themajor signaling pathways that induce M2 gene expression) was evaluatedby Western blot. In the pathway, Akt phosphorylation occurs in responseto an upstream signal, and 4EBP1 phosphorylation and S6K phosphorylationfurther occur through mTORC1 in the downstream. The S6K phosphorylationprovides a negative feedback to the upstream of the signaling pathway.Western blot was performed as in Example 5-2, except that IL-4 (20ng/ml), methylcobalamin, and RAD001 (200 nM) were added at 30 minutesbefore the proteins were collected, and the primary antibodies fordetection were changed.

The results are shown in FIGS. 8-1, 8-2, and 8-3 . The valuescorresponding to data in the graphs of FIGS. 8-1, 8-2, and 8-3 are shownin Table 8. When IL-4 was added, activation of Akt and both 4EBP1 andS6K in the downstream was observed. As compared with IL-4 alone, theactivation of 4EBP1 and the activation of S6K were both enhanced whenIL-4 and 100 nM methylcobalamin were used in combination. However, whenthe methylcobalamin concentration was 1 mM, the activity of Akt in theupstream was decreased although 4EBP1 and S6K were activated. WhenRAD001, which is an inhibitor of mTOR, was further added in addition toIL-4 and methylcobalamin, the activity of Akt in the upstream wasrescued, and the activities of 4EBP1 and S6K in the downstream weresuppressed. Thus, a mechanism was suggested in which the Akt activity toinduce M2 gene in the upstream was suppressed by a negative feedbackmechanism from the downstream when a high concentration ofmethylcobalamin was added.

TABLE 8 IL-4 − + + + + MeCbl − − 100 nM 1 mM 1 mM RAD001 − − − − +p-Akt/ Average 1.00 2.05 3.58 2.59 3.78 Akt Standard 0.04 0.14 0.21 0.190.19 error Standard 0.07 0.24 0.37 0.32 0.34 deviation p-4EBP1/ Average1.00 2.12 4.94 6.08 2.78 4EBP1 Standard 0.12 0.37 0.25 0.34 0.58 errorStandard 0.21 0.63 0.43 0.58 1.01 deviation p-S6K/ Average 1.00 1.802.65 3.12 0.33 S6K Standard 0.15 0.02 0.06 0.09 0.13 error Standard 0.260.04 0.11 0.16 0.23 deviation

Example 7. Analysis of Macrophage Phenotypes after Sciatic Nerve Injury

Effects of methylcobalamin on macrophage phenotypes after sciatic nerveinjury were analyzed by an immunohistological evaluation method.Specifically, the analysis was carried out as follows. Using transversenerve sections at 2.5 mm in the proximal direction, at the injury site,and at 2.5, 5.0, and 7.5 mm in the distal direction, macrophages wereevaluated by immunofluorescence staining at 1, 3, 7, and 14 days aftersciatic nerve injury. The proximal direction refers to a cell body sideof an axon with respect to an injury site, and the distal directionrefers to a terminal side of an axon with respect to an injury site.Each of the distances indicates a distance from the injury site (thesame applies to Example 8). Macrophages were labeled with CD68, an M1marker iNOS, and an M2 marker CD206. The proportion of M1 macrophage wascalculated by the following formula: Proportion of M1 macrophage(%)=number of M1-marker positive macrophages per mm²/number ofmacrophages per mm²×100. A more detailed description will be providedbelow.

<Example 7-1. Surgical Treatment (Rat Sciatic Nerve Crush Injury Model)>

Six-week-old male Wistar rats (about 200 g) were used. The left sciaticnerve was exteriorized, and crush injury of the sciatic nerve was madeon the proximal side by forceps. The nerve was crushed for 10 secondsthree times at 10-second intervals. The fascia and skin were suturedusing 3-0 nylon. A non-injury group which underwent only sciatic nerveexposure, an untreated group, and a methylcobalamin treatment group werecompared. To administer methylcobalamin continuously, an osmoticminipump was placed and left in the dorsal subcutaneous space, andmethylcobalamin was administered at a dose of 1 mg/kg/day. In anuntreated group, physiological saline was administered instead ofmethylcobalamin according to the same procedure.

<Example 7-2. Morphological and Histological Analysis>

At 1, 3, 7, and 14 days after the surgery, rats were anesthetized withan anesthetic agent. The left sciatic nerve was collected, fixed with 4%PFA for 7 days and with 20% sucrose for 24 hours, and the fixed nervewas freeze-embedded. The embedded tissue was sliced into a 5-μm-thickslice in a nerve short-axis direction and placed on a glass slide.Slices were obtained from 5 positions of 2.5 mm in the proximaldirection from an injured region, the injured region, and 2.5, 5.0, and7.5 mm in the distal direction. The slices were dried for 1 hour andfixed with 95% methanol for 30 minutes. The fixed slices were blockedand allowed to react with primary antibodies at 4° C. overnight. Thecells were further allowed to react with a secondary antibody at roomtemperature for 1 hour so that the nuclei were labeled with DAPI.

The primary antibodies used were a mouse anti-CD68 monoclonal antibody(Abcam), a rabbit anti-iNOS monoclonal antibody (Abcam), a rabbitanti-CD206 monoclonal antibody (Abcam), a rabbit anti-neurofilament 200(NF 200) polyclonal antibody (SIGMA), and a mouse anti-myelin basicprotein (MBP) monoclonal antibody (Calbiochem). The secondary antibodiesused were an Alexa 488-labeled goat anti-mouse IgG antibody (LifeTechnologies), an Alexa 488-labeled goat anti-rabbit IgG antibody (LifeTechnologies), an Alexa 568-labeled goat anti-mouse IgG antibody (LifeTechnologies), and an Alexa 568-labeled goat anti-rabbit IgG antibody(Life Technologies).

<Example 7-3. Results>

The results are shown in FIGS. 9 and 10 . The values corresponding todata in the graphs of FIG. 9 are shown in Tables 9-1, 9-2, and 9-3, andthe values corresponding to data in the graphs of FIG. 10 are shown inTables 10-1, 10-2, and 10-3. In the methylcobalamin treatment group, thenumbers of macrophages accumulated in the injury site were significantlydecreased at 3, 7, and 14 days after the surgery, as compared to theuntreated group. At the distal positions, the number of macrophages wasincreased behind the injury site, and there was a significant differenceat 14 days after the surgery.

In the methylcobalamin treatment group, the numbers of M1 macrophageswere significantly decreased at all evaluation days. In the distalportions, there were significant differences at 7 and 14 days after thesurgery. Similar trends were observed for the proportions of M1macrophages.

In the methylcobalamin treatment group, the numbers of M2 macrophageswere significantly increased at 1, 7 and 14 days after the surgery atthe injury site. There was a significant difference at 5 mm in thedistal direction at 7 days after the surgery and at 7.5 mm in the distaldirection at 7 and 14 days after the surgery. Similar trends wereobserved for the proportions of M2 macrophage.

TABLE 9-1 Number of macrophages 2.5 mm in the 2.5 mm in 5.0 mm in 7.5 mmin proximal the distal the distal the distal direction Injury sitedirection direction direction POD1 CTR Average 40.14 418.55 75.56 62.2045.38 Standard error 5.58 22.81 8.25 5.69 6.94 Standard deviation 13.6755.87 20.22 16.38 16.99 MeCbl Average 40.53 335.86 64.37 43.67 35.68Standard error 3.47 32.55 11.81 6.02 3.84 Standard deviation 8.50 78.7428.92 14.74 9.42 Sham Average 12.56 17.12 19.41 18.65 13.70 Standarderror 5.71 1.74 6.04 6.12 1.32 Standard deviation 9.89 3.02 10.46 10.612.28 POD3 CTR Average 81.25 1672.85 209.11 143.85 158.68 Standard error13.14 125.76 33.95 28.74 39.69 Standard deviation 46.88 308.04 83.1770.40 37.23 MeCbl Average 48.52 1261.77 181.38 135.88 124.99 Standarderror 5.34 112.29 25.25 26.07 19.03 Standard deviation 13.07 275.0661.85 63.87 46.63 Sham Average 20.55 22.07 18.65 16.74 17.12 Standarderror 13.46 13.35 11.15 8.70 10.15 Standard deviation 23.31 23.12 19.3215.07 17.57 POD7 CTR Average 74.92 1402.02 994.51 985.01 856.04 Standarderror 22.81 45.54 74.84 154.53 115.89 Standard deviation 55.88 111.78183.31 378.66 283.87 MeCbl Average 36.63 1096.37 921.92 763.17 584.14Standard error 5.30 60.32 50.32 43.99 39.33 Standard deviation 12.38147.76 123.26 122.45 96.33 Sham Average 7.51 7.61 15.50 11.42 9.51Standard error 1.37 3.63 4.03 5.23 4.39 Standard deviation 2.38 6.296.98 3.06 7.60 POD14 CTR Average 67.04 1327.81 1447.08 1187.11 989.31Standard error 27.39 106.88 102.42 82.54 70.54 Standard deviation 67.09259.36 250.87 202.19 172.78 MeCbl Average 51.79 899.57 898.81 827.51752.23 Standard error 5.77 50.94 61.50 50.46 12.32 Standard deviation14.14 124.77 150.63 123.59 30.18 Sham Average 12.18 14.46 9.13 9.1316.74 Standard error 5.53 5.98 2.38 2.38 7.72 Standard deviation 9.5710.36 4.12 4.12 13.38

TABLE 9-2 Number of M1 macrophages 2.5 mm in the 2.5 mm in 5.0 mm in 7.5mm in proximal the distal the distal the distal direction Injury sitedirection direction direction POD1 CTR Average 1.90 123.29 16.74 10.856.50 Standard error 0.70 21.41 6.37 4.51 2.15 Standard deviation 1.7252.44 15.60 11.06 5.28 MeCbl Average 3.42 63.55 10.27 4.40 3.21 Standarderror 0.51 6.55 2.48 0.64 1.25 Standard deviation 1.25 15.04 6.08 1.583.08 Sham Average 1.52 0.00 0.76 1.52 1.52 Standard error 1.52 0.00 0.761.52 0.76 Standard deviation 2.54 0.00 1.32 2.64 1.32 POD3 CTR Average6.09 379.02 58.44 32.14 28.10 Standard error 0.96 42.23 17.81 9.07 7.77Standard deviation 2.36 103.44 43.63 22.22 13.02 MeCbl Average 3.04138.36 23.50 13.13 15.57 Standard error 1.13 33.23 4.12 3.15 3.19Standard deviation 2.76 81.33 10.09 7.72 7.80 Sham Average 2.28 0.000.76 0.76 0.00 Standard error 1.32 0.00 0.76 0.76 0.00 Standarddeviation 2.28 0.00 1.32 1.32 0.00 POD7 CTR Average 5.33 109.25 79.65102.58 108.44 Standard error 2.51 18.99 13.48 14.38 23.24 Standarddeviation 6.40 46.51 33.01 35.23 56.94 MeCbl Average 1.54 47.56 37.0925.29 22.71 Standard error 0.77 7.92 8.97 5.44 2.75 Standard deviation1.88 19.40 21.97 13.32 5.73 Sham Average 0.76 1.52 2.28 0.75 0.76Standard error 0.76 0.76 1.32 0.76 0.76 Standard deviation 1.32 1.322.28 1.32 1.32 POD14 CTR Average 6.09 69.10 91.97 87.58 61.97 Standarderror 2.93 8.70 8.82 16.23 13.23 Standard deviation 7.17 21.32 21.5039.75 32.40 MeCbl Average 2.34 21.33 27.86 22.35 15.59 Standard error0.83 2.10 5.32 2.34 2.88 Standard deviation 2.04 5.14 13.04 5.72 7.06Sham Average 0.76 0.76 0.76 0.76 0.00 Standard error 0.76 0.76 0.76 0.760.00 Standard deviation 1.32 1.32 1.32 1.32 0.00

TABLE 9-3 M1 macrophage ratio 2.5 mm in the 2.5 mm in 5.0 mm in 7.5 mmin proximal the distal the distal the distal direction Injury sitedirection direction direction POD1 CTR Average 0.084 0.262 0.183 0.1430.152 Standard error 0.029 0.038 0.050 0.040 0.027 Standard deviation0.071 0.092 0.121 0.098 0.066 MeCbl Average 0.095 0.195 0.183 0.1090.097 Standard error 0.019 0.012 0.041 0.024 0.030 Standard deviation0.048 0.030 0.100 0.058 0.073 POD3 CTR Average 0.088 0.226 0.200 0.1920.190 Standard error 0.013 0.016 0.038 0.026 0.030 Standard deviation0.031 0.040 0.094 0.064 0.073 MeCbl Average 0.068 0.163 0.165 0.1450.186 Standard error 0.025 0.020 0.033 0.029 0.025 Standard deviation0.061 0.049 0.081 0.071 0.061 POD7 CTR Average 0.085 0.079 0.080 0.1040.126 Standard error 0.022 0.014 0.011 0.016 0.039 Standard deviation0.054 0.034 0.026 0.038 0.096 MeCbl Average 0.049 0.046 0.043 0.0390.045 Standard error 0.022 0.009 0.009 0.009 0.007 Standard deviation0.055 0.022 0.023 0.022 0.017 POD14 CTR Average 0.082 0.051 0.066 0.0770.062 Standard error 0.010 0.004 0.010 0.015 0.012 Standard deviation0.023 0.011 0.024 0.036 0.029 MeCbl Average 0.039 0.025 0.033 0.0290.024 Standard error 0.013 0.004 0.004 0.003 0.004 Standard deviation0.031 0.009 0.009 0.007 0.009

TABLE 10-1 Number of macrophages 2.5 mm in the 2.5 mm in 5.0 mm in 7.5mm in proximal the distal the distal the distal direction Injury sitedirection direction direction POD1 CTR Average 40.14 418.55 75.56 62.2045.38 Standard error 5.58 22.81 8.25 6.69 6.34 Standard deviation 13.6755.87 20.22 16.38 16.99 MeCbl Average 40.53 336.86 64.37 43.67 35.68Standard error 3.47 32.55 11.81 6.02 3.84 Standard deviation 8.50 73.7428.92 14.74 3.42 Sham Average 12.56 17.12 19.41 18.65 13.70 Standarderror 5.71 1.74 6.04 6.12 1.32 Standard deviation 9.89 3.02 10.46 10.612.28 POD3 CTR Average 81.25 1672.85 208.11 143.85 158.68 Standard error19.14 125.76 33.95 28.74 39.69 Standard deviation 46.88 308.04 83.1770.40 97.23 MeCbl Average 48.52 1261.77 181.38 135.88 124.99 Standarderror 5.34 112.29 25.25 26.07 19.00 Standard deviation 13.07 275.0661.85 63.87 46.63 Sham Average 20.55 22.07 18.65 16.74 17.12 Standarderror 13.46 13.35 11.15 8.70 10.15 Standard deviation 23.31 23.12 19.3215.07 17.57 POD7 CTR Average 74.32 1402.02 994.51 986.01 856.04 Standarderror 22.81 45.64 74.84 154.59 115.89 Standard deviation 55.88 111.78183.31 378.66 283.87 MeCbl Average 36.63 1096.37 921.92 763.17 584.14Standard error 5.20 60.32 50.32 49.99 39.33 Standard deviation 12.98147.76 123.26 122.45 96.33 Sham Average 7.61 7.61 15.60 11.42 9.51Standard error 1.37 3.63 4.00 5.23 4.39 Standard deviation 2.38 5.296.98 9.06 7.60 POD14 CTR Average 57.04 1327.81 1447.08 1187.11 389.31Standard error 27.39 105.88 102.42 82.54 70.54 Standard deviation 57.03259.35 250.97 202.13 172.78 MeCbl Average 51.79 899.57 898.81 827.51752.29 Standard error 5.77 50.94 61.50 50.46 12.32 Standard deviation14.14 124.77 150.63 123.59 30.18 Sham Average 12.18 14.46 9.13 9.1316.74 Standard error 5.53 5.98 2.38 2.38 7.72 Standard deviation 9.5710.36 4.12 4.12 13.38

TABLE 10-2 Number of M2 macrophages 2.5 mm in the 2.5 mm in 5.0 mm in7.5 mm in proximal the distal the distal the distal direction Injurysite direction direction direction POD1 CTR Average 38.05 97.45 49.3045.86 27.28 Standard error 10.42 13.34 10.28 10.30 9.54 Standarddeviation 25.53 34.14 25.14 25.22 23.36 MeCbl Average 32.83 138.28 45.5333.90 32.83 Standard error 6.28 5.65 14.10 7.73 5.27 Standard deviation15.39 13.84 34.53 18.94 12.91 Sham Average 8.37 7.61 19.79 11.42 6.85Standard error 4.03 2.74 10.24 7.34 2.64 Standard deviation 6.69 4.7517.73 12.71 4.57 POD3 CTR Average 54.73 542.53 99.63 96.94 112.25Standard error 15.76 64.42 28.47 19.34 35.57 Standard deviation 38.61157.80 69.75 71.89 87.36 MeCbl Average 43.76 603.12 147.74 117.16 114.48Standard error 9.59 45.84 25.64 26.59 23.23 Standard deviation 23.48112.28 62.80 65.12 57.06 Sham Average 8.37 7.61 9.89 8.37 5.85 Standarderror 7.26 5.33 5.49 4.53 2.64 Standard deviation 12.57 9.23 8.51 8.024.57 POD7 CTR Average 31.83 489.91 339.75 320.70 252.81 Standard error10.84 36.40 46.66 13.69 14.25 Standard deviation 26.56 89.17 113.5033.53 34.91 MeCbl Average 26.25 654.63 457.16 432.13 327.16 Standarderror 8.06 51.64 21.88 39.46 21.91 Standard deviation 19.73 126.49 53.6036.66 53.66 Sham Average 3.81 4.57 6.85 6.08 6.09 Standard error 2.013.49 3.95 6.08 3.81 Standard deviation 3.49 6.04 6.85 10.55 6.59 POD14CTR Average 29.13 303.34 303.53 343.53 274.42 Standard error 8.77 55.7454.74 53.15 33.10 Standard deviation 21.49 136.52 134.08 130.20 81.07MeCbl Average 35.65 560.45 443.91 434.68 417.63 Standard error 6.1336.00 46.74 49.13 26.20 Standard deviation 15.02 88.19 114.50 120.3564.18 Sham Average 6.08 4.57 7.61 5.33 12.18 Standard error 4.03 3.491.52 2.74 7.61 Standard deviation 6.98 6.04 2.64 4.75 13.18

TABLE 10-3 M2 macrophage ratio 2.5 mm in the 2.5 mm in 5.0 mm in 7.5 mmin proximal the distal the distal the distal direction Injury sitedirection direction direction POD1 CTR Average 0.678 0.267 0.624 0.7160.705 Standard error 0.063 0.026 0.033 0.035 0.057 Standard deviation0.153 0.063 0.081 0.087 0.139 MeCbl Average 0.743 0.431 0.658 0.7650.777 Standard error 0.057 0.059 0.028 0.024 0.041 Standard deviation0.140 0.130 0.068 0.060 0.100 POD3 CTR Average 0.608 0.320 0.548 0.7040.682 Standard error 0.046 0.012 0.048 0.036 0.024 Standard deviation0.112 0.030 0.116 0.089 0.058 MeCbl Average 0.708 0.479 0.693 0.6590.694 Standard error 0.027 0.037 0.023 0.021 0.025 Standard deviation0.066 0.090 0.055 0.050 0.062 POD7 CTR Average 0.408 0.350 0.399 0.3830.370 Standard error 0.032 0.024 0.032 0.018 0.015 Standard deviation0.079 0.059 0.079 0.044 0.036 MeCbl Average 0.642 0.571 0.463 0.5050.506 Standard error 0.027 0.019 0.031 0.032 0.021 Standard deviation0.066 0.047 0.076 0.079 0.050 POD14 CTR Average 0.534 0.249 0.228 0.2890.287 Standard error 0.037 0.056 0.050 0.043 0.040 Standard deviation0.091 0.138 0.123 0.107 0.097 MeCbl Average 0.698 0.610 0.459 0.4870.487 Standard error 0.045 0.031 0.042 0.032 0.032 Standard deviation0.110 0.077 0.102 0.078 0.078

Example 8. Analysis of Nerve Regeneration after Sciatic Nerve Injury

Effects of methylcobalamin on nerve regeneration after sciatic nerveinjury were analyzed by an immunohistological evaluation method.Specifically, the analysis was carried out as follows. Transverssections of an injured sciatic nerve obtained at 2 weeks after sciaticnerve injury were used for evaluation. As in the macrophage evaluation,evaluations were carried out for the sections at 2.5 mm in the proximaldirection from an injury site, the injury site, and 2.5, 5.0, and 7.5 mmin the distal direction. Regenerated axons were labeled with NF 200, andmyelin sheaths were labeled with MBP. The myelination rate of theregenerated axons was calculated by the following formula: Myelinationrate (%)=number of myelinated axons per mm²/number of axons per mm²×100.More specifically, the analysis was performed in the same manner as inExample 7.

The results are shown in FIG. 11 . The values corresponding to data inthe graphs of FIG. 11 are shown in Tables 11-1, 11-2, and 11-3. In themethylcobalamin treatment group, the numbers of axons and the numbers ofmyelinated axons were significantly improved at the injury site. Therewere significant differences in the numbers of axons at 5.0 and 7.5 mmin the distal direction, in the number of myelinated axons at 2.5, 5.0,and 7.5 mm in the distal direction, and in the myelination rate at 5.0and 7.5 mm in the distal direction. These results and the results inExample 7 indicated that methylcobalamin promotes nerve regeneration inan anti-inflammatory manner by decreasing M1 macrophages and increasingM2 macrophages in an actual nerve regeneration process.

TABLE 11-1 Number of axons 2.5 mm in the 2.5 mm in 5.0 mm in 7.5 mm inproximal the distal the distal the distal direction Injury sitedirection direction direction POD14 CTR Average 17797 11202 13130 111309278 Standard error 821 329 243 279 306 Standard deviation 2010 805 596684 750 MeCbl Average 16251 14210 14478 16663 14678 Standard error 783843 500 945 666 Standard deviation 1917 2064 1224 2316 1632 Sham Average17432 18923 18137 19267 19455 Standard error 649 472 434 22 670 Standarddeviation 1123 818 751 38 1160

TABLE 11-2 Number of myelinated axons 2.5 mm in the 2.5 mm in 5.0 mm in7.5 mm in proximal the distal the distal the distal direction Injurysite direction direction direction POD14 CTR Average 17263 6601 52571306 608 Standard 767 470 383 244 66 error Standard 1879 1150 938 599162 deviation MeCbl Average 15838 9785 7620 4400 2473 Standard 767 748869 1139 596 error Standard 1878 1832 2128 2789 1461 deviation ShamAverage 17019 18377 17663 18698 18815 Standard 655 475 422 19 626 errorStandard 1135 824 731 32 1084 deviation

TABLE 11-3 Myelination ratio 2.5 mm in the 2.5 mm in 5.0 mm in 7 5 mm inproximal the distal the distal the distal direction Injury sitedirection direction direction POD14 CTR Average 0.971 0.592 0.401 0.1180.066 Standard 0.007 0.045 0.029 0.022 0.008 error Standard 0.016 0.1110.071 0.055 0.020 deviation MeCbl Average 0.975 0.691 0.531 0.253 0.163Standard 0.002 0.042 0.064 0.055 0.033 error Standard 0.005 0.104 0.1560.135 0.080 deviation Sham Average 0.976 0.971 0.974 0.970 0.967Standard 0.004 0.001 0.002 0.002 0.003 error Standard 0.006 0.002 0.0030.003 0.005 deviation

Example 9. Therapeutic Effect on Spinal Cord Injury

Therapeutic effects of methylcobalamin on spinal cord injury wereexamined by Basso-Beattie-Bresnahan (BBB) score and thermal algesimetrytest. Specifically, the examination was carried out as follows.

<Example 9-1. Construction of Rat Spinal Cord Injury Model (LateralHemisection Model) and Drug Administration>

Six-week-old female Wistar rats were used. The rats were purchased fromCharles River Laboratories Japan, Inc. (Yokohama, Japan). Anesthesia wasperformed as follows. A 1:10 dilution of a mixture of three anestheticagents in physiological saline was administered by intraperitonealinjection. One dose consisted of 0.2 mg/kg of midazolam, 0.015 mg/kg ofmedetomidine, and 0.25 mg/kg of butorphanol. The rat was placed in aprone position on an operating table, and a dorsal midline incision wasmade. The T10 vertebral arch was removed to expose the posterior face ofthe spinal cord, and the left spinal cord was hemisected using asharp-pointed surgical scalpel (spitz mess). The skin was sutured usinga 4-0 nylon thread and the surgery was completed. A comparison was madeamong three groups: a methylcobalamin treatment group, an untreatedgroup, and a sham group. Immediately after the surgical operation, anosmotic minipump filled with methylcobalamin (1 mg/kg/day) orphysiological saline was placed for the methylcobalamin treatment groupand the untreated group, respectively, and left in the left dorsalsubcutaneous space. The sham group underwent only resection of the Th10vertebral arch.

<Example 9-2. Measurement of BBB Score>

Each rat was separately accommodated in a cage, allowed to walk freely,and observed for 5 minutes. According to a conventional method,left-lower-extremity function was evaluated using scores of 0 (nolocomotion) to 21 (normal locomotion). The evaluations were performedbefore surgery, and at 1, 7, 14, 21, and 28 days after the surgery.

<Example 9-3. Thermal Algesimetry Test>

Each rat was separately accommodated in a dedicated cage, an infraredthermal stimulation was applied to the right sole, and time until therat retracted the hind limb was measured. To avoid damage to the skin,the stimulation was continued for 15 seconds at the maximum. Theevaluations were performed before surgery, and at 7, 14, 21, and 28 daysafter the surgery.

<Example 9-4. Results>

BBB scores are shown in FIG. 12 . The values corresponding to data inthe graph of FIG. 12 are shown in Table 12. As compared to the untreatedgroup, the left-lower-extremity motor function was significantlyimproved in the methylcobalamin administration group at 14, 21, and 28days after the surgery.

TABLE 12 BBB score Number of days elapsed after a surgery 0 1 7 14 21 28CTR Average 21 0 3.17 9 10.33 10.5 Standard 0 0 1.64 2.42 2.80 2.86error Standard 0 0 4.02 5.93 6.86 7.01 deviation MeCbl Average 21 0.178.33 15.50 16.33 17 Standard 0 0.17 2.20 0.67 0.67 0.58 error Standard 00.41 5.39 1.64 1.63 1.41 deviation Sham Average 21 20.8 21 21 21 21Standard 0 0.2 0 0 0 0 error Standard 0 0.45 0 0 0 0 deviation

The results of the thermal algesimetry test are shown in FIG. 13 . Thevalues corresponding to data in the graph of FIG. 13 are shown in Table13. In the methylcobalamin treatment group, the right-lower-extremityhyperesthesia was significantly improved at 21 and 28 days after thesurgery.

TABLE 13 Thermal algesimetry test Time after spying infrared thermalstimulation Number of days elapsed after a surgery 0 7 14 21 28 CTRAverage 9.85 6.64 7.26 6.97 7.54 Standard 0.37 0.33 0.41 0.37 0.26 errorStandard 0.91 0.82 1.01 0.90 0.64 deviation MeCbl Average 9.99 8.59 8.869.61 9.92 Standard 0.48 0.55 0.53 0.40 0.34 error Standard 1.18 1.361.30 0.99 0.84 deviation Sham Average 9.87 9.62 9.41 9.75 9.50 Standard0.48 0.21 0.23 0.14 0.22 error Standard 1.07 0.47 0.51 0.32 0.49deviation

Example 10. M2 Microglia Induction Promoting Effect and M1 MicrogliaInduction Inhibiting Effect

An M2 microglia induction promoting effect and an M1 microglia inductioninhibiting effect of methylcobalamin were examined by a Western blotmethod. Specifically, the examination was carried out as follows.

<Example 10-1. Western Blot>

To a microglia cell line (6-3 cells), LPS (100 ng/ml) and ananti-inflammatory cytokine IL-4 (20 ng/ml) were added. Methylcobalaminsolutions each having a different concentration ranging from 1 nM to 1mM were added to the mixture, and proteins were recovered from eachmixture at 1 and 3 days after the addition. Electrophoresis and transferto a membrane were performed, the membrane was blocked, and the cellswere allowed to react with each of primary antibodies against M1 markers(iNOS and IL-1β) and M2 markers (Arg1 and CD206) at 4° C. overnight.Cells were allowed to react with a secondary antibody at roomtemperature for 1 hour, and bands were detected using a detector.

The primary antibodies used were an anti-iNOS antibody, an anti-IL-1(3antibody, an anti-Arg1 antibody, and an anti-CD206 (a mannose receptor)antibody. The secondary antibody used was an Anti-Rabbit IgG, HRP-LinkedWhole Ab Sheep.

<Example 10-2. Results>

The results of Western blot are shown in FIGS. 14 to 15 . The valuescorresponding to data in the graphs of FIG. 14 are shown in Table 14,and the values corresponding to data in the graphs of FIG. 15 are shownin Table 15. The amounts of pro-inflammatory (M1) markers of microgliawere as follows: as compared with the case LPS alone, the amount ofIL-1β protein was significantly decreased when at least 1 μMmethylcobalamin was added in combination, and the amount of iNOS proteinwas significantly decreased when at least 10 nM methylcobalamin wasadded in combination. The amounts of anti-inflammatory (M2) markers wereas follows: as compared with IL-4 alone, the amount of Arg1 protein wassignificantly increased when 1 nM to 10 μM methylcobalamin was added incombination. The amount of CD206 was significantly increased when 10 nMto 100 nM methylcobalamin was added in combination.

TABLE 14 LPS − + + + + + + + + MeCbl − − 1 nM 10 nM 100 nM 1 μM 10 μM100 μM 1 mM IL-1β Average 0.06 1.00 1.02 0.95 0.83 0.72 0.58 0.53 0.32Standard 0.01 0.06 0.06 0.04 0.04 0.06 0.09 0.06 0.04 error Standard0.01 0.10 0.11 0.07 0.06 0.10 0.10 0.10 0.06 deviation INOS Average 0.171.00 0.87 0.74 0.76 0.61 0.46 0.36 0.05 Standard 0.09 0.10 0.02 0.040.02 0.06 0.07 0.04 0.04 error Standard 0.15 0.17 0.03 0.07 0.03 0.100.13 0.07 0.06 deviation

TABLE 15 IL-4 − + + + + + + + + MeCbl − − 1 nM 10 nM 100 nM 1 μM 10 μM100 μM 1 mM Arg1 Average 0.13 1.00 1.78 2.03 2.72 2.20 2.08 1.37 0.59Standard 0.04 0.19 0.30 0.13 0.10 0.08 0.02 0.13 0.20 error Standard0.06 0.32 0.52 0.23 0.18 0.14 0.04 0.22 0.34 deviation CD206 Average0.63 1.00 1.11 1.42 1.39 1.17 1.17 1.24 1.11 Standard 0.01 0.06 0.050.03 0.11 0.01 0.06 0.19 0.07 error Standard 0.02 0.10 0.09 0.05 0.190.01 0.10 0.33 0.12 deviation

Example 11. M2 Macrophage Induction Promoting Effect and M1 MacrophageInduction Inhibiting Effect

An M2 macrophage induction promoting effect and an M1 macrophageinduction inhibiting effect of methylcobalamin were examined.Specifically, the examination was carried out as follows.

<Example 11-1. Immunofluorescent Staining>

The rat spinal cord injury model of Example 9-1 was used. At 7, 14, and28 days after the surgery, the rats were anesthetized with an anestheticagent, and perfusion fixation was performed with 4% PFA. Then, thespinal cord including the injury site was collected and fixed with 20%sucrose for 24 hours, and the fixed spinal cord was freeze-embedded. Theembedded tissue was sliced into a 5-μm-thick slice in a nerve short-axisdirection and placed on a glass slide. The slice was dried for 1 hourand fixed with 100% methanol for 30 minutes. The fixed slices wereblocked, and cells were allowed to react with primary antibodies at 4°C. overnight. The cells were further allowed to react with a secondaryantibody at room temperature for 1 hour so that the nuclei were labeledwith DAPI.

Using transverse spinal cord sections on the affected side at 2 and 1 mmon the head side and at 1 and 2 mm on the tail side from the injurysite, the number of macrophages, the number of M1 (pro-inflammatorytype) macrophages, the proportion of M1 macrophages, the number of M2(anti-inflammatory type) macrophages, and the proportion of M2macrophage per unit area, as well as the M1/M2 ratio were measured andcalculated.

The primary antibodies used were an anti-CD68 antibody, an anti-iNOSantibody, and an anti-Arg1 antibody. The secondary antibodies used werean Alexa 488-labeled goat anti-rabbit IgG antibody and an Alexa568-labeled goat anti-mouse IgG antibody.

<Example 11-2. Results>

The results of the immunofluorescent staining are shown in FIGS. 16 to22 . FIGS. 16 to 18 show differences in the number of macrophages, thenumber of M1 (pro-inflammatory type) macrophages, the proportion of M1macrophages, the number of M2 (anti-inflammatory type) macrophages, andthe proportion of M2 macrophages per unit area for each position. FIGS.19 to 20 show changes with time in days after the surgery. FIG. 21 showschanges in the M1/M2 ratio with time in days after the surgery. FIG. 22shows changes at each position. The values corresponding to data in thegraphs of FIGS. 16 to 18 are shown in Tables 16 to 18, respectively, andthe values corresponding to data in the graphs of FIG. 22 are shown inTable 19.

In the methylcobalamin treatment group, there were trends for a smallernumber of accumulated macrophages per unit area as compared with theuntreated group. Further, in view of the phenotypes of the macrophages,there were trends for decreased M1 macrophages and increased M2macrophages. There were significant differences in some results.

TABLE 16 Number of macrophages 2 mm on the 1 mm on the 1 mm on the 2 mmon the head side head side tail side tail side OTR Average 301.82 573.09736.03 461.60 Standard deviation 168.61 167.02 177.69 186.35 MeCblAverage 143.68 266.62 348.57 155.09 Standard deviation 54.14 112.44101.19 68.10 Number of M1 macrophages/mm2 2 mm on the 1 mm on the 1 mmon the 2 mm on the head side head side tail side tail side OTR Average65.06 130.79 166.83 97.60 Standard deviation 21.28 32.98 74.57 40.46MeCbl Average 20.43 37.00 43.07 27.02 Standard deviation 7.99 21.74 9.7012.54 Number of M2 macrophages/mm2 2 mm on the 1 mm on the 1 mm on the 2mm on the head side head side tail side tail side OTR Average 37.7383.66 80.48 32.27 Standard deviation 19.78 28.17 36.41 19.79 MeCblAverage 25.87 63.85 76.13 28.76 Standard deviation 7.71 27.22 29.2211.21 M1 macropage ratio 2 mm on the 1 mm on the 1 mm on the 2 mm on thehead side head side tail side tail side OTR Average 0.240 0.231 0.2220.208 Standard deviation 0.091 0.035 0.058 0.049 MeCbl Average 0.1430.131 0.129 0.170 Standard deviation 0.021 0.032 0.031 0.025 M2macrophage ratio 2 mm on the 1 mm on the 1 mm on the 2 mm on the headside head side tail side tail side OTR Average 0.128 0.152 0.120 0.063Standard deviation 0.027 0.050 0.026 0.021 MaCbl Average 0.193 0.2420.219 0.194 Standard deviation 0.068 0.053 0.045 0.050

TABLE 17 Number of macrophages 2 mm on the 1 mm on the 1 mm on the 2 mmon the head side head side tail side tail side OTR Average 398.46 693.35617.71 399.81 Standard deviation 100.33 156.42 165.81 182.93 MeCblAverage 271.15 362.17 395.70 254.39 Standard deviation 163.51 166.70146.05 173.16 Number of M1 macropages/mm2 2 mm on the 1 mm on the 1 mmon the 2 mm on the head side head side tail side tail side OTR Average53.70 100.09 95.75 54.77 Standard deviation 24.77 29.96 66.00 55.25MeCbl Average 28.35 48.35 43.74 25.54 Standard deviation 19.78 40.6130.34 20.78 Number of M2 macrophages/mm2 2 mm on the 1 mm on the 1 mm onthe 2 mm on the head side head side tail side tail side OTR Average31.07 52.81 42.74 24.18 Standard deviation 17.65 33.23 19.84 18.80 MeCblAverage 36.37 47.90 43.12 28.29 Standard deviation 29.10 32.79 16.997.84 M1 macrophage ratio 2 mm on the 1 mm on the 1 mm on the 2 mm on thehead side head side tail side tail side OTR Average 0.129 0.144 0.1430.120 Standard deviation 0.030 0.029 0.072 0.057 MeCbl Average 0.1030.122 0.102 0.094 Standard deviation 0.017 0.050 0.038 0.020 M2macrophage ratio 2 mm on the 1 mm on the 1 mm on the 2 mm on the headside head side tail side tail side OTR Average 0.076 0.075 0.072 0.059Standard deviation 0.039 0.041 0.037 0.040 MeCbl Average 0.146 0.1300.112 0.143 Standard deviation 0.080 0.043 0.034 0.086

TABLE 18 Number of macrophages 2 mm on the 1 mm on the 1 mm on the 2 mmon the head side head side tail side tail side OTR Average 318.74 41.455439.96 378.94 Standard error 26.40 15.21 31.15 36.07 Standard deviation54.66 37.25 76.31 86.35 MeCbl Average 230.75 320.22 342.11 246.52Standard error 35.06 30.83 48.83 25.69 Standard deviation 85.89 75.51119.60 66.36 Number of M1 macrophages/mm2 2 mm on the 1 mm on the 1 mmon the 1 mm on the head side head side tail side tail side OTR Average33.93 51.06 64.73 50.43 Standard error 2.11 3.53 9.30 6.35 Standarddeviation 5.16 8.64 22.77 15.55 MeCbl Average 20.15 29.78 44.79 24.58Standard error 2.95 6.03 8.94 6.03 Standard deviation 7.23 14.93 21.9114.77 Number of M2 macrophages/mm 2 2 mm on the 1 mm on the 1 mm on the2 mm on the head side head side tail side tail side OTR Average 4.2811.76 10.14 4.40 Standard error 0.89 2.11 1.43 0.50 Standard deviation2.17 5.17 3.51 1.23 MeCbl Average 7.65 12.86 10.33 7.42 Standard error2.11 2.76 1.15 0.89 Standard deviation 5.17 6.81 2.81 2.19 M1 macrophageratio 2 mm on the 1 mm on the 1 mm on the 2 mm on the head side headside tail side tail side OTR Average 0.106 0.124 0.151 0.133 Standarderror 0.007 0.003 0.023 0.007 Standard deviation 0.016 0.023 0.057 0.017MeCbl Average 0.069 0.090 0.132 0.095 Standard error 0.005 0.012 0.0170.015 Standard deviation 0.013 0.028 0.042 0.007 M2 macropahge ratio 2mm on the 1 mm on the 1 mm on the 2 mm on the head side head side tailside tail side OTR Average 0.014 0.028 0.024 0.012 Standard error 0.0040.005 0.004 0.001 Standard deviation 0.009 0.011 0.011 0.000 MeCblAverage 0.032 0.009 0.002 0.002 Standard error 0.006 0.006 0.000 0.006Standard deviation 0.014 0.013 0.007 0.014

TABLE 19 M1/M2 2 mm on the 1 mm on the 1 mm on the 2 mm on the head sidehead side tail side tail side 7 days CTR Average 1.98230737 1.7371791.87175346 3.62357386 after a Standard 1.01770554 0.8941413 0.447867171.63595842 surgery deviation MeCbl Average 0.81556762 0.57223070.60197181 0.93373695 Standard 0.28148608 0.20701435 0.14455330.30181695 deviation 14 days CTR Average 2.11618015 2.877832532.61877661 3.35640625 after a Standard 1.17059363 2.18023676 1.91279023.17882907 surgery deviation MeCbl Average 0.91059534 0.9930490.94086406 0.86048838 Standard 0.48325852 0.36363151 0.381032270.47425723 deviation 28 days CTR Average 11.6333333 5.291093477.46137087 11.8402778 after a Standard 10.0675947 2.73818316 4.321163533.17208053 surgery deviation MeCbl Average 3.35546661 2.456545484.29967382 3.54549168 Standard 2.01009777 0.82739767 1.541863832.36798272 deviation

Example 12. Function Recovery Promoting Effect of Methylcobalamin inPhotocoagulation-induced Cerebral Infarction Model

<Example 12-1. Subject and Method>

Eight to 10-week-old male C57BL/6J mice (about 24 g) were used. Aphotocoagulation-induced cerebral infarction model for laser lightirradiation after rose bengal administration was constructed. In thismodel, the skull of each mouse was drilled to open a hole having acenter at 2 mm from the anterior fontanel in the outward direction. At 5minutes after a photosensitive dye rose bengal was administered, theright motor cortex as a center was irradiated with laser light to createcerebral infarction in the right lateral motor cortex. An osmotic pump(ALZET osmotic pumps, for 2-week operation) was embedded immediatelyafter the creation of the cerebral infarction. The mice were dividedinto a methylcobalamin treatment group (N=3) and an untreated group(N=4), and rotarod tests (accelerating velocity method) were carried outat 2, 4, 7, 9, 11, and 14 days after the surgery creating cerebralinfarction. The time until a mouse fell from a rotarod was measured, anda ratio to a maximum of 300 seconds as baseline was calculated.

<Example 12-2. Results>

The results are shown in FIG. 23 . The value corresponding to data inthe graph of FIG. 23 are shown in Table 20. At 2, 4, and 9 days afterthe surgery creating cerebral infarction, brain function examined in therotarod test significantly improved in the methylcobalamin treatmentgroup as compared with the untreated group.

TABLE 20 After PIT operation 0 day 2 days 4 days 7 days 9 days 11 days14 days CTR Average 0.998 0.101 0.144 0.263 0.208 0.322 0.368 Standarddeviation 0.003 0.021 0.070 0.115 0.069 0.175 0.198 MeCbl Average 0.9980.470 0.573 0.370 0.437 0.521 0.510 Standard deviation 0.004 0.020 0.0480.024 0.155 0.062 0.017

The invention claimed is:
 1. A method of promoting M2macrophage/microglia induction, inhibiting M1 macrophage/microgliainduction, and/or reducing the ratio of M1 macrophage/microglia to M2macrophage/microglia in a patient in need thereof, the method comprisingadministering an agent comprising an effective amount of vitamin B₁₂ tothe patient to promote M2 macrophage/microglia induction, inhibit M1macrophage/microglia induction, and/or reduce the ratio of M1macrophage/microglia to M2 macrophage/microglia.
 2. The method accordingto claim 1, wherein the vitamin B₁₂ is at least one selected from thegroup consisting of methylcobalamin, cyanocobalamin, hydroxocobalamin,sulfitocobalamin, adenosylcobalamin, and salts thereof.
 3. The methodaccording to claim 1, wherein the vitamin B₁₂ is methylcobalamin.
 4. Themethod according to claim 1, wherein the agent is administered to thepatient continuously for a period of time.
 5. The method according toclaim 1, wherein the agent is administered to the patient by intravenousdrip infusion.